The current project continues to focus on a transcription factor called CREB3L1, which is synthesized as a membrane-bound precursor and activated through a process known as regulated intramembrane proteolysis (RIP). The protein contains a single transmembrane helix, with the N-terminal domain facing the cytosol. During the last grant cycle we have determined that TGF- induces cleavage of CREB3L1, allowing the N-terminal domain of the protein to enter nucleus where it activates transcription of genes stimulating assembly of collagen- containing extracellular matrix. Since TGF--induced excess deposition of the collagen- containing matrix leads to tissue fibrosis, inhibiting proteolytic activation of CREB3L1 may be useful in treating fibrotic diseases. This hypothesis will be tested in Aim 1 of the proposal in which we will determine the roles of CREB3L1 in obesity-induced fibrosis of adipose tissue using mice in which CREB3L1 is selectively ablated in adipocytes. Achieving this aim may determine whether proteolytic activation of CREB3L1 could be a novel drug target to treat lipotoxicity by inhibiting fibrosis of adipose tissue. In addition to TGF-,we have determined that doxorubicin also stimulates cleavage of CREB3L1, allowing the N-terminal domain of the protein to activate genes that inhibit cell proliferation. We demonstrated that doxorubicin blocked proliferation of cancer cells through activating RIP of CREB3L1. This observation led us to propose Aim 2 in which we will determine whether CREB3L1 expression may serve as a biomarker for doxorubicin- based chemotherapy. Achieving this aim will markedly improve the response rate of doxorubicin by allowing identification of patients who are likely to benefit from the drug treatment. We have further determined that doxorubicin activates CREB3L1 cleavage by inducing synthesis of ceramide. A crucial step for ceramide to activate cleavage of CREB3L1 is to invert the membrane orientation of a transmembrane protein called TM4SF20 by blocking the insertion of its signal peptide into membranes. We designate this novel regulatory mechanism as alternative translocation. Aim 3 of the project is proposed to delineate the mechanism through which transmembrane and secretory proteins are regulated by alternative translocation. Achieving this aim will demonstrate how membrane proteins can adopt different membrane topologies, and how secretory proteins can function intracellularly under certain physiological conditions. This study should profoundly broaden our views to these proteins.